The ability to cultivate parasites in vitro has resulted in rapid progress in the understanding of host-parasite relationships, parasite biology and pathophysiology. Conversely, the lack of a parasite in vitro culture system can severely hamper studies on that specific pathogen as seen in Babesia microti-caused babesiosis. This is true despite the mounting evidence for the emergence and spread of B. microti in the US and its dominating status as a threat to transfusion medicine. The overall goal of our proposal is to establish a continuous in vitro culture system of B. microti in human RBCs to enable a platform amenable for future experimental investigation. It is well known that human RBCs which serve as the cellular vehicle host for Babesia are not a uniform, homogenous population of cells in circulation and undergo many age related transformations in their 120 day life span. Our hypothesis is that these heterogeneous RBCs are not equal hosts for B. microti invasion and growth. The two specific aims in our proposal will test this hypothesis and assess if parasite invasion and proliferation in human RBCs would occur when presented with a uniform RBC population of the optimum age and features. Specific Aim 1 addresses the identification of specific human RBC sub-populations that would support optimum invasion and replication of B. microti in vitro and Specific Aim 2 will define culture conditions (gas, media, shaking vs static) needed to support this invasion and replication of B. microti in vitro. By combining our extensive experience in parasitology (Lobo and Narla, P. falciparum and B. divergens) together with the red cell expertise (Narla) and reagents (Human sources of B. microti, umbilical cord cell resources, antibodies, and antigens) available at NYBC, we will obtain insights into the specific RBC lineage favored by B. microti. This study is the first systematic analysis of culture conditions required for parasite propagation and is a necessary first step in the search for reagents for diagnosis, epidemiology, treatment and prevention of human babesiosis, as well as to assist in developing novel methods to screen blood products.